Viral hemorrhagic fevers (VHFs) constitute a group of severe illnesses in which the vascular system is damaged with accompanying internal bleeding, while regulatory functions of the body are critically impaired. Several distinct families of viruses cause VHF, with varying disease severity. The most dangerous VHF, associated with a mortality as high as 90%, is caused by the filoviruses (Filoviridae), such as Marburg virus (MARV) and Ebola virus (EBOV). Although outbreaks of Ebola hemorrhagic fever, first identified in 1976, are sporadic and endemic to Africa, EBOV constitutes a grave global potential health threat.
Five strains of EBOV have been identified to date. These include four African strains, including the Tai Forest (also known as Ivory Coast), Sudan, Zaire, and Bundibugyo, as well as the Reston strain from the Philippines. There is only one known MARV strain that is related to the EBOV strains. In addition, the distantly related Lloviu virus (LLOV) is found in insectivorous bats in Spain. Reston EBOV and Lloviu virus do not appear to be pathogenic in humans. In contrast, the Sudan, Ivory Coast, Bundibugyo and Zaire EBOV strains, as well as the MARV strain, have been associated with human VHF outbreaks.
Fatality rates for the EBOV viruses may range from about 20% to about 90%. For example, the EBOV ranges from about 40% for the Sudan and Bundibugyo strains to about 90% for the Zaire strain. The rate for the Tai Forest strain is not yet known owing to its rarity.
The highly pathogenic Marburg and Ebola viruses (MARV and EBOV) were discovered in 1967 and 1976, respectively. Although both cause deadly hemorrhagic fever with 20-90% mortality, until recently they caused only sporadic outbreaks confined to Central Africa. The endemic nature of these outbreaks provided limited incentive for development of effective vaccines, therapeutics and diagnostics.
The situation changed dramatically in 2014, when the world was confronted with the first widespread Ebola epidemic. This was the most challenging EBOV outbreak ever reported by the World Health Organization affecting people in Guinea and Liberia, with possible cases in Sierra Leone, Mali, and Ghana. There were also isolated cases identified in Spain, United States, United Kingdom and Italy. This was a specter of a worldwide epidemic.
Although the African outbreak has been mostly contained, as of July 2015 more than 27,000 suspected, probable and confirmed cases have been reported, with more than 11,000 documented deaths (CDC website). These figures most likely underestimate the real spread of the epidemic. The outbreak is still ongoing in Guinea and Sierra Leone, and a new case was diagnosed in Liberia even after the World Health Organization declared it free of the disease.
Owing to its pathogenicity, high mortality, and human-to-human transmission, EBOV and MARV are considered to be potential bioweapons and are classified as a Category A bioterrorism agent. Importantly, there are no approved vaccines or antiviral agents against EBOV, while existing therapies for infected individuals have minimal effects. The unprecedented severity of the 2014/2015 EBOV fever outbreak underscores the clear and present danger posed by the virus.
Considerable effort and resources have now been invested in the fight against EBOV, and rapid progress is being made towards development and approval of therapeutic antibodies, vaccines, and diagnostic tools. Of special note is the preparation of the recombinant ZMAPP therapeutic antibodies, which were shown to reverse the Ebola fever in non-human primates. These antibodies are currently undergoing clinical trials launched by the NIH in a partnership with the Liberian government. Further, there is considerable hope that an effective vaccine may soon be available: an rVSV-vectored vaccine expressing the Zaire EBOV surface glycoprotein has undergone an interim randomized trial with very promising results.
Nevertheless, much remains to be done. Currently, there are no small molecule drugs targeting the EBOV or MARV viruses, and more importantly, there are no inexpensive, reliable point-of-care diagnostics. Since it is unrealistic to expect that the whole population of Central and West Africa will be vaccinated any time soon, it is imperative that cheap and reliable diagnostic tools are developed. In particular, tools are necessary so that whenever a potentially infected individual is identified, we can quickly confirm the presence of EBOV or, even better, identify the viral strain. For example, there is intense interest in the molecular mechanisms of infectivity, replication, assembly, and pathogenesis of EBOV in humans, with the long-term objective of identifying suitable targets for drug discovery and the development of effective diagnostic tools.
A target of EBOV diagnostic and therapeutic research is the EBOV ssRNA genome. The EBOV ssRNA genome encodes seven proteins, most of which have multiple functions. Two of the EBOV proteins are the glycoprotein (GP) and the matrix protein, VP40, which are essential components of the viral envelope that surrounds the nucleocapsid. The nucleocapsid includes a viral negative-sense ssRNA complexed with five additional proteins, such as the Nucleoprotein or Nucleoprotein (NP), the VP24, VP30 and VP35 structural proteins, and the viral polymerase (L).
Nucleoproteins (NP) are found in all members of the order Mononegavirales, which groups together a number of important viruses that are highly pathogenic to humans, animals and plants, including Filoviridae, measles, mumps and rabies viruses, avian bornavirus, and many others. The ssRNA in these viruses is packaged into a helical complex that includes multiple copies of NP. The architectures of the resulting NP-ssRNA complexes differ among the Mononegavirales families. Insights into the structure-function relationships underlying the physiological role of NPs from Mononegavirales have been made possible owing to crystallographic studies of the proteins from rabies virus and bomavirus.
For example, Cryo-EM and tomography allowed for the reconstruction of the EBOV nucleocapsid at about 14 Angstrom (Å) to about 19 Å resolution. Recent intensive efforts have resulted in structural characterization, often using crystallography, of the five EBOV proteins, GP, VP40, VP24, VP30, and VP35. Proteins L and NP have so far eluded structural characterization. The NP plays a critical role in virus replication and maturation, and is the most abundant viral protein in infected cells and the viral nucleocapsid.
Interestingly, the Filoviridae members appear to have unusual NPs characterized by a longer polypeptide chain than those of other Mononegavirales, with two distinct functional modules, and the N-terminal domain exhibiting the canonical ssRNA-packaging function. Recent data suggest that the C-terminal domain, with an amino-acid sequence that shows no homology to any other protein, may serve as a unique hub for protein-protein interactions in the nucleocapsid that are distinct from any other Mononegavirales. Moreover, recent data show that the C-terminal fragment of the EBOV NP is a major antigenic determinant, raising the possibility that it could be effective in virus detection and diagnostics.
Rapid diagnostics make it possible to confirm or discard cases at points of treatment, reduce danger of infections in non-EBOV-positive patients, guide triage and clinical care. Until recently, the only approved diagnostic tool for EBOV detection was a PCR test which takes 2-6 hours, was contingent on access to proper instrumentation, and costs approximately $100 per assay. The World Health Organization (WHO) recently called for a test suited for use in peripheral health clinics with no access to laboratory infrastructure, taking no more than two steps with results no later than in 30 minutes, and with biosafety requirements limited to the use of personal protective equipment. Ideally, such inexpensive tests should selectively recognize EBOV antigens with high sensitivity specific in bodily fluids. Such diagnostic tests should also provide rapid, direct readout in a manner similar to fertility tests based in general terms on ELISA technology. Prototypes of such kits, made by the European companies Vedalab and Senova, are currently being tested in West Africa.
On Mar. 16, 2015 the ReEBOV test produced by Corgenix Medical Corp. became the first test to be approved for EBOV diagnosis, albeit in an emergency situation. A field validation study of the ReEBOV test was recently published. The ReEBOV test is a chromatographic immunoassay designed for qualitative detection of VP40, one of the seven viral proteins which are synthesized in the infected cells in addition to the glycoprotein (GP), nucleoprotein (NP), the L-polymerase, VP24, VP30 and VP35.
The ReEBOV assay uses affinity purified, polyclonal antibody obtained from goats immunized with a recombinant VP40 antigen. Whole blood or plasma from individuals suspected of EBOV infection is used in the test that is conducted as a dipstick immunoassay. If present in the sample, the EBOV VP40 antigen forms complexes with the anti-EBOV VP40 antibody conjugated to gold nanoparticles. This step generates a pink to red signal and provides a visual positive readout.
In an immunoassay, the detection antibody is responsible for the sensitivity, specificity, and/or selectivity of the assay. The full production cycle of polyclonal antibodies from goats, which are used when large amounts of antiserum is required, is approximately eighty (80) days. However, polyclonal IgG antibodies cannot be produced by cell lines and must be produced each time in the animal. The amounts of antibodies and their specificity and selectivity cannot be predicted or modified in a rational way. Polyclonal antibodies are also relatively unstable.
For example, ReEBOV kits should be kept at 4° C. to keep the antibody stable, and thus, requires a cold transport chain. Antibody production, storage, and transport costs are a significant portion of the cost associated with any diagnostic kit, including the ReEBOV kits. Nonetheless, the results of a recent Lancet published validation study and an independent WHO study differ significantly, but both studies suggest a significant level of false positive readouts are obtained by the ReEBOV kits.
Described herein is an innovative approach which uses proven technology to overcome virtually all of the limitations that stem from the use of natural polyclonal antibodies in diagnostic, therapeutic, or research kits. The present disclosure describes replacement of polyclonal antibodies with recombinant, synthetic Fragment antigen-binding proteins (Fabs) generated using a phage display system. This technology offers numerous advantages over the canonical production of polyclonal antibodies. In particular, the present disclosure describes the elucidation and manipulation of the molecular architecture of the EBOV Nucleoprotein as a significant advancement in the efforts to aid the development of recombinant Fab antibodies directed to bind to the EBOV NP antigen. The antibodies described herein provide a key mechanism to begin to effectively treat the EBOV epidemic in West Africa and other effected parts of the world.